Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITIONS AND METHODS FOR PRODUCING STABLE VIRAL VECTOR
PRODUCER CELLS FOR CELL AND GENE THERAPY
CROSS-REFERENCE TO RELATED APPLICATIONS
[001] This application claims priority from U. S . Provisional Patent
Application No.
63/025,812, filed May 15, 2020, which is herein incorporated by reference in
its entirety.
FIELD
[002] The present disclosure relates to the field of the production of
viral vectors for cell
and gene therapy.
BACKGROUND
[003] The growing number of gene therapy candidates combined with rapid
progression
through the clinical development has created a world-wide shortage of gene
therapy vectors.
More than 500 gene therapy and 100 cell therapy candidates are in different
stages of
development. Greater than 2200 clinical studies are ongoing across the globe.
The strong and
proven safety profile of viral vectors (e.g., lentiviral vectors) has
underpinned a robust clinical
development pipeline. However, the clinical manufacture and use of viral
vectors, especially
lentiviral vectors, also comes with several limitations.
For example, conventional
manufacturing methods and associated technologies are outdated and not
scalable, provide low
downstream process yields (-20%), and furthermore require significant upfront
capital and
ongoing operational costs to establish. Furthermore, traditionally, viral
vector manufacturing
is seen as unpredictable and highly risky, resulting in demand greatly
exceeding supply, which
in turn drives up prices. There is a need to identify new methods and
improvement for
manufacturing viral vectors by generating stable producer lines with high
titer at high volumes.
SUMMARY
[004]
In an aspect, the present disclosure provides a method of making a stable
viral
vector producer cell line, the method comprising:
a.
introducing into a population of cells a viral vector genome construct
encoding
a gene of interest (GOT) and one or more viral accessory constructs encoding
one or
more viral accessory proteins;
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b. producing a population of transgenic cells comprising integrated or
episomal
sequences encoding the GOT and the one or more viral accessory proteins;
c. selecting from the population of transgenic cells a cell clone producing
a
desired viral titer; and
d. generating from the cell clone a stable viral vector producer cell line,
wherein the introduction of the one or more accessory constructs occurs
concurrently.
[005] In another aspect, the present disclosure provides a method of
making a stable viral
vector producer cell line, the method comprising:
a. introducing into a population of cells a viral vector genome construct
encoding
a gene of interest (GOT) and one or more viral accessory constructs encoding
one or
more viral accessory proteins;
b. producing a population of transgenic cells comprising integrated or
episomal
sequences encoding the GOT and the one or more viral accessory proteins;
c. selecting from the population of transgenic cells a cell clone producing
a
desired viral titer; and
d. generating from the cell clone a stable viral vector producer cell line,
wherein the introduction of the one or more accessory constructs occurs via
one or more sequential steps with no intervening cell culturing.
BRIEF DESCRIPTION OF THE DRAWINGS
[006] Figure 1 provides an illustration of the genome organization of the
HIV-1 Virus.
The HIV-1 genome contains 9,749 bp. In addition to the gag, poi, and env genes
common to
all retroviruses, HIV-1 contains a regulatory gene - rev - that is
indispensable for virus
replication, and five accessory genes - vif, vpr, vpu, tat, and nef - that,
while dispensable for
in vitro virus growth, are key for in vivo replication and pathogenesis.
Further information
about the biological functions of each of the HIV-encoded proteins is provided
in Table 1.
[007] Figure 2 provides an exemplary work flow of generating a cell clone
with stable
introduction of various construct elements.
[008] Figure 3 provides exemplary vector constructs used in Example 2.
[009] Figure 4 provides results of the Example 2 experiments showing
optimal conditions
for GFP (combinations 16 and 4) and optimal conditions for Globin-LCR-GFP
(combinations
12 and 6).
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DETAILED DESCRIPTION
[010] Unless defined otherwise, technical and scientific terms used herein
have the same
meaning as commonly understood by one of ordinary skill in the art. One
skilled in the art will
recognize many methods can be used in the practice of the present disclosure.
Indeed, the
present disclosure is in no way limited to the methods and materials
described. Where a term
is provided in the singular, the inventors also contemplate aspects of the
disclosure described
by the plural of that term, and vice versa. Where there are discrepancies in
terms and
definitions used in references that are incorporated by reference, the terms
used in this
application shall have the definitions given herein. Other technical terms
used have their
ordinary meaning in the art in which they are used, as exemplified by various
art-specific
dictionaries, for example, "The American Heritage Science Dictionary"
(Editors of the
American Heritage Dictionaries, 2011, Houghton Mifflin Harcourt, Boston and
New York),
the "McGraw-Hill Dictionary of Scientific and Technical Terms" (6th edition,
2002, McGraw-
Hill, New York), or the "Oxford Dictionary of Biology" (6th edition, 2008,
Oxford University
Press, Oxford and New York).
[011] Any references cited herein, including, e.g., all patents and
publications are
incorporated by reference in their entirety.
[012] When a grouping of alternatives is presented, any and all
combinations of the
members that make up that grouping of alternatives is specifically envisioned.
For example, if
an item is selected from a group consisting of A, B, C, and D, the inventors
specifically envision
each alternative individually (e.g., A alone, B alone, etc.), as well as
combinations such as A,
B, and D; A and C; B and C; etc. The term "and/or" when used in a list of two
or more items
means any one of the listed items by itself or in combination with any one or
more of the other
listed items. For example, the expression "A and/or B" is intended to mean
either or both of A
and B ¨ i.e., A alone, B alone, or A and B in combination. The expression "A,
B and/or C" is
intended to mean A alone, B alone, C alone, A and B in combination, A and C in
combination,
B and C in combination, or A, B, and C in combination.
[013] When a range of numbers is provided herein, the range is understood
to inclusive
of the edges of the range as well as any number between the defined edges of
the range. For
example, "between 1 and 10" includes any number between 1 and 10, as well as
the number 1
and the number 10.
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[014] As used herein, the singular form "a," "an," and "the" include plural
references
unless the context clearly dictates otherwise. For example, the term "a
compound" or "at least
one compound" may include a plurality of compounds, including mixtures thereof
[015] The term "about" is used herein to mean approximately, roughly,
around, or in the
region of. When the term "about" is used in conjunction with a numerical
range, it modifies
that range by extending the boundaries above and below the numerical values
set forth, and is
understood to mean plus or minus 10%. For example, "about 100" would include
from 90 to
110.
[016] As used herein, the term "substantially", when used to modify a
quality, generally
allows certain degree of variation without that quality being lost. For
example, in certain
aspects such degree of variation can be less than 0.1%, about 0.1%, about
0.2%, about 0.3%,
about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about
1%, between
1-2%, between 2-3%, between 3-4%, between 4-5%, or greater than 5%.
[017] To avoid any doubt, used herein, terms or phrases such as "about",
"at least", "at
least about", "at most", "less than", "greater than", "within" or "alike",
when followed by a
series of list of numbers of percentages, such terms or phrases are deemed to
modify each and
every number of percentage in the series or list, regardless whether the
adverb, preposition, or
other modifier phrase is reproduced prior to each and every member.
[018] As used herein, a "viral vector producer cell" refers to a cell which
contains all the
.. elements necessary for production of recombinant viral vector particles
(including e.g.,
retroviral delivery systems). Typically, such viral vector producer cell
contains one or more
expression cassettes which are capable of expressing viral structural proteins
(such as gag, pol
and env). A "stable viral vector producer cell" refers to a viral vector
producer cell that contains
in its nuclear genome, maintains episomally, or combination thereof, all the
elements necessary
.. for production of recombinant viral vector particles. A "stable viral
vector producer cell line"
refers to a permanently established cell culture of stable viral vector
producer cells that will
proliferate indefinitely given appropriate fresh medium and space.
[019] As used herein, a "recombinant viral vector" is an enveloped virion
particle that
contains an expressible polynucleotide sequence, and which is capable of
penetrating a target
host cell, thereby carrying the expressible sequence into the cell. In an
aspect, an expressible
polynucleotide sequence comprises or encodes a gene of interest (GOT). The
enveloped
particle is preferably pseudotyped with an engineered or native viral envelope
or capsid protein
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from another viral species, including lentiviruses or non-lentiviruses, which
alters the host
range and infectivity of a native virus.
[020] As used herein, a "viral vector genome construct" is a construct
which contains
polynucleotide sequences which are packaged into a transducing recombinant
viral vector. In
an aspect, a viral vector genome construct, when comprising 5' LTR and 3' LTR
and packaged
with a functional integrase enzyme, can be used for the production of
recombinant viral vectors
that are capable of integrating into the host genome. In another aspect, a
viral vector genome
construct produces a recombinant viral vector comprising 5' LTR and 3' LTR and
not capable
of integrating into a host genome due to the lack of a functional integrase
enzyme, which is
also known as an integrase-defective lentiviral vector (IDLV).
[021] As used herein, a "viral accessory construct" refers to a construct,
plasmid or
isolated nucleic acid molecule containing or encoding one or more elements
that are useful for
producing a functional recombinant viral vector in a compatible host cell, and
packaging into
it an expressible heterologous sequence.
[022] As used herein, a "viral vector construct" refers to either a viral
vector genome
construct or a viral accessory construct.
[023] As used herein, the term "operably linked" describes the spatial
relationship of two
or more pieces of DNA such that one piece is capable of effecting an intended
genetic outcome
of another piece. For example, "operably linked" can denote a relationship
between a
regulatory region (typically a promoter element, but may include an enhancer
element) and the
coding region of a gene, whereby the transcription of the coding region is
under the control of
the regulatory region.
[024] As used herein, a "concatemer" is defined as a continuous DNA
molecule that
contains multiple copies of the same or substantially same DNA sequence linked
in series. In
an aspect, a concatemer may also contain one or more selection genes.
[025] As used herein, the term "trans" refers to mechanisms acting from
different
molecules.
[026] As used herein, the term "promoter" includes nucleic acid regions
ranging in
complexity and size from minimal promoters to promoters including upstream
elements and
enhancers.
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[027] As used herein, the term "transduction" refers to the delivery of a
nucleic acid
segment using a viral vector by means of viral vector.
[028] As used herein, the term "transfection" refers to the introduction of
foreign DNA
into eukaryotic cells.
[029] Without being bound to any theory, quality and quantity of infectious
vector
particles derived from a viral vector producer cell line is directly affected
by the stoichiometric
ratio of the lentiviral vector genomic RNA to the trans expressed accessory
proteins. For any
given lentiviral vector genome, the optimal ratio is not known a priori, and
must be determined
empirically through trial and error. As this biological fact is not often
appreciated, the
construction of stable cell lines has historically been accomplished by the
addition of accessory
genes one at a time in a serial fashion. This has assured progeny clones that
had and expressed
the accessory protein but limited the ability of the ultimate cell line to
produce vector for
lentiviral vector genomes with suboptimal ratios. The solution offered to this
problem is to add
all of the accessory elements at once in such a manner as to encourage
multiple introductions
of each of the elements. This not only speeds the development time of any
given producer clone
by collapsing the accessory gene introductions from multiple rounds of
subcloning to a single
round, it also allows for the generation of a diverse set of clones, each with
different ratios,
such that when the clones are screened the likelihood that we can find a clone
that produces
vector of the desired quality and quantity is increased without having to know
a priori what
that ratio would be.
[030] In an aspect, this disclosure provides a method to achieve natural
selection of
optimized stable vector producing cell lines by random assortment using
shotgun cloning. In
an aspect, this application provides a stable lentiviral vector producer cell
line provided by
introducing both a lentiviral vector genome as well as lentiviral accessory
proteins expressed
in trans from separately introduced constructs.
[031] In an aspect, a vector producer cell line is produced from a parental
cell line derived
from an immortalized human cell line. In another aspect, a vector producer
cell line grows in
defined media either with or without human/animal derived serum. In another
aspect, a vector
producer cell line grows in an adherent or suspension adapted manner.
Recombinant viral vectors
[032] This disclosure relates to the manufacturing and/or production of
recombinant viral
vectors (also known as recombinant viral particles). The present disclosure
relates to
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recombinant viral vectors, and constructs for their manufacture, which can be
utilized to
introduce expressible polynucleotide sequences of interest into host cells.
[033] In an aspect, a viral vector producer cell disclosed herein comprises
a retroviral
production system, wherein the viral vector is derived from a retrovirus.
Retroviruses comprise
a family of enveloped viruses with a 7-12kb single-stranded positive sense RNA
genome. The
retrovirus family includes five groups of oncogenic retroviruses, lentiviruses
and
spumaviruses.
[034] Retroviral vector production systems typically involve separation of
viral genome
from viral packaging functions. Viral accessory proteins or viral accessory
protein domains
may be introduced via separate expression cassettes, or in trans. In an
aspect, a viral accessory
construct encodes or provides one or more viral accessory proteins involved in
viral packaging.
[035] In an aspect, the present disclosure relates to lentiviral vectors,
and constructs for
their manufacture, which can be utilized to introduce expressible
polynucleotide sequences of
interest into host cells. In an aspect, a lentiviral vector is an enveloped
virion particle that
contains an expressible polynucleotide sequence, and which is capable of
penetrating a target
host cell, thereby carrying the expressible sequence into the cell. The
enveloped particle is
preferably pseudotyped with an engineered or native viral envelope protein
from another viral
species, including non-lentiviruses, which alters the host range and
infectivity of the native
lentivirus.
[036] Viral vectors described here can be utilized in a wide range of
applications,
including, e.g., for protein production (including vaccine production), for
gene therapy
(including gene replacement, gene editing, and synthetic biology), to deliver
therapeutic
polypeptides, to deliver siRNA, ribozymes, anti-sense, and other functional
polynucleotides,
etc. Such transduction vectors have the ability to carry single or dual genes,
and to include
inhibitory sequences (e.g., RNAi or antisense). In certain aspects, the
transduction vector also
carries a nucleic acid which comprises a modified 3' LTR having reduced, but
not absent,
transcriptional activity.
[037] Lentivirus is a group of retroviruses characterized for a long
incubation period.
They are classified into five serogroups according to the vertebrate hosts
they infect: bovine,
equine, feline, ovine/caprine and primate. Some examples of lentiviruses are
human (HIV),
simian (SIV) and feline (FIV) immunodeficiency viruses.
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[038] Lentiviruses can deliver large amounts of genetic information into
the DNA of host
cells and can integrate in both dividing and non-dividing cells. The viral
genome is passed
onto daughter cells during division, making it one of the most efficient gene
delivery vectors.
[039] The structure of HIV is different from that of other retroviruses.
HIV is roughly
spherical with a diameter of ¨120 nm. HIV is composed of two copies of
positive ssRNA that
code for nine genes enclosed by a conical capsid containing 2,000 copies of
the p24 protein.
The ssRNA is tightly bound to nucleocapsid proteins, p'7, and enzymes needed
for the
development of the virion: Reverse transcriptase (RT), Proteases (PR),
Ribonuclease and
Integrase (IN). A matrix composed of p17 surrounds the capsid ensuring the
integrity of the
virion. This, in turn, is surrounded by an envelope composed of two layers of
phospholipids
taken from the membrane of a human cell when a newly formed virus particle
buds from the
cell. Embedded in the viral envelope are proteins from the host cell and about
70 copies of a
complex HIV protein, known as Env, that protrudes through the surface of the
virus particle.
Env consists of a cap made of three gp120 molecules, and a stem consisting of
three gp41
molecules that anchor the structure into the viral envelope. The glycoprotein
complex enables
the virus to attach to and fuse with target cells to initiate the infectious
cycle. Further
information about the biological functions of each of the HIV-encoded proteins
is provided in
Table 1.
Table 1: Summary of the biological functions of HIV-encoded proteins.
Gene
Precursor proteins products
gag Group-specific antigen gag
4 MA, CA, SP1, N. SP2, P6
poi Polymerase poi 4 RT, RNase H, IN,
PR
Essential Genes for
Vectorized Lentivirus env Envelope gp160 4 gp120, gp41
rev Regulator of expression of
important for major viral protein synthesis
virion proteins and
essential for viral replication
tat HIV transact ivator
Positive transcription regulator
vif Viral infectivity
Required for infectivity in some cell types
Nuclear import of pre-integration complex
Additional Genes found in vpr Virus protein R
and host cell cycle arrest
Wild-Type HIV
Proteasomal degradation of CD44 and virion
vpu Virus protein U
release from infected cells
nef Negative factor Roles in apoptosis and
virus infectivity
[040] In an aspect, a viral vector producer cell disclosed herein comprises
a lentiviral
vector production system, wherein the viral vector is derived from a
lentivirus. A lentivirus is
a group of retroviruses that causes slow, gradual disease. A lentiviral vector
particle produced
by the lentiviral vector production system disclosed herein will be capable of
transducing
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slowly-dividing cells, whereas standard retroviruses (gamma retroviruses) can
infect only
mitotically active cells. "Slowly dividing" cell types may divide
approximately once every
three to four days.
[041] In the production of lentiviral vectors, multiple plasmids are used,
one encoding
envelope proteins (env plasmid), one or more plasmids encoding viral accessory
proteins, and
one plasmid comprising a gene of interest expression cassette between a
lentiviral 3'-LTR and
a lentiviral 5'-LTR to facilitate integration of the encoded gene(s) of
interest into the host
genome.
[042] In an aspect, a viral vector may be a hybrid viral vector. The term
"hybrid" as used
herein refers to a vector, or nucleic acid component of a vector, that
contains both lentiviral
sequences and non-lentiviral sequences.
[043] In an aspect, a viral vector producer cell disclosed herein comprises
a herpesvirus
vector production system, wherein the viral vector is derived from a
herpesvirus.
[044] In an aspect, a viral vector producer cell disclosed herein comprises
an adenoviral
vector production system, wherein the viral vector is derived from an
adenovirus. Adenovirus
is a nonenveloped virus with a 36-kilobase double-stranded DNA genome.
Adenovirus is an
attractive gene delivery vehicle candidate for its ability to grow as a high-
titer recombinant
virus, large transgene capacity, and efficient transduction of dividing and
non-dividing cells.
More than 50 human and nonhuman serotypes of adenovirus have been found to
mediate gene
delivery to a wide range of tissues.
[045] In an aspect, a viral vector producer cell disclosed herein comprises
an adeno-
associated viral vector production system, wherein the viral vector is derived
from an adeno-
associated virus. Adeno-associated virus (AAV) is a nonenveloped virus with a
4.7kb single-
stranded DNA genome. More than 100 serotypes of AAV have been isolated from
human and
nonhuman tissues.
[046] In a further aspect, a recombinant viral vector disclosed herein is
derived from a
virus comprising a mosaic genome structure. In a further aspect, recombinant
viral vectors
disclosed herein are target-specific. In a further aspect, target-specific
viral vectors are
receptor-targeted. In a further aspect, target-specific viral vectors comprise
recombinant
antibody molecules. Methods to produce target-specific viral vectors are known
in the art. In
a further aspect, a recombinant vector is derived from a partially or fully
synthetic nucleic acid
sequence.
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[047] Recombinant viral vectors disclosed herein may have one or more
selectable,
traceable or otherwise detectable marker elements. In an aspect, a selectable
element is a
reporter gene. In a further aspect, a selectable element is an epitope tag. In
a further aspect, a
viral vector may contain both a reporter gene and an epitope tag. In an
aspect, an epitope tag
may be selected or detected by methods known in the art, including but not
limited to
chromatography, enzyme assays, fluorescence assays, and immunodetection
assays. In an
aspect, immunodetection assays may include, but are not limited to
immunoblotting,
immunofluorescence, immunocytochemistry, and enzyme-linked immunosorbent assay
(ELISA).
[048] In a further aspect, a reporter gene may be detected by methods to
detect
absorbance. Methods to detect absorbance are known in the art. In an aspect, a
reporter gene
may be detected by methods to detect fluorescence. Methods to detect
fluorescence are known
in the art. In a further aspect, a reporter gene may be detected by methods to
detect
luminescence. Methods to detect luminescence are known in the art. In an
aspect, a selectable
.. marker gene is an antibiotic resistance gene. In a further aspect, an
antibiotic gene is encodes
neomycin resistance. In a further aspect, an antibiotic gene encodes puromycin
resistance.
[049] In a further aspect, traceable marker genes may include genes
encoding fluorescent
proteins. Methods to select fluorescent proteins with different chromophores
are known in the
art. In a further aspect, fluorescent proteins may be green fluorescent
protein (GFP) or variants
thereof, including, but not limited to Ultramarine, blue and cyan fluorescent
proteins. In a
further aspect, a variant of a fluorescent protein may be an optimized
variant. Methods to
optimize traits of fluorescent proteins are known in the art and include, but
are not limited to
methods to improve chromophore maturation, folding kinetics, and
thermostability, among
other traits.
[050] In an aspect, a recombinant viral vector may be self-inactivating.
The terms "self-
inactivating" refer to a vector which is modified, such that the modification
reduces the ability
of the vector to mobilize once it has integrated into the genome of a target
or host cell. For
example, the modification may include deletions in the 3' long terminal repeat
(LTR) region.
SIN vectors possess safety advantages over non-SIN vectors for gene delivery
applications.
[051] In another aspect, a recombinant viral vector produced here is a Self-
Inactivating
Lentiviral Vectors (SIN vectors). In a SIN vector, the deletion of lentiviral
enhancer and
promoter sequences from the 3' LTR results in the generation of vectors which,
on infection of
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target cells, are incapable of transcribing vector-length RNA. Because of this
modification,
integrated SIN vectors are incapable of further replication thus reducing the
likelihood of
generating replication-competent viruses as well as the danger of
inadvertently influencing
transcription activity of nearby endogenous promoters.
[052] In another aspect, a recombinant viral vector produced here is a
conditional SIN
vector. For example, in an exemplary conditional SIN vector, the 3' LTR U3
transcription
regulatory elements can be replaced with an inducible promoter (e.g., Tet-
responsive element).
Viral vector genome construct
[053] In the disclosure disclosed herein, a viral vector genome construct
encodes a gene
of interest. In an aspect, a gene of interest is operably linked to a
promoter.
[054] In an aspect, a gene of interest may be a candidate gene which is of
known or
potential significance in the pathophysiology of a disease. In a further
aspect, a gene of interest
may have a known or potential therapeutic or diagnostic application. In an
aspect, a gene of
interest may comprise a coding region. In a further aspect, a gene of interest
may comprise a
.. partial coding region. A gene of interest can be obtained for insertion
into the viral vectors
disclosed herein through a variety of techniques known in the art.
[055] In a further aspect, a viral vector genome construct disclosed herein
comprises one
or more selectable or detectable element(s). In an aspect, a selectable or
detectable element is
a reporter. In a further aspect, a selectable or detectable aspect is an
epitope tag. In an aspect,
a selectable or detectable elements may be selected or detected by methods
known in the art
including, but not limited to luminescence, absorbance, fluorescence,
antibiotics, antigen-
antibody interactions, or a combination thereof.
[056] In an aspect, a viral vector genome construct disclosed herein
comprises one or
more elements selected from the group consisting of a promoter, 5' and 3' long
terminal
.. repeats, a packaging signal, a central polypurine tract, and a
polyadenylation sequence (p(A)).
In another aspect, a viral vector genome construct disclosed herein comprises
all the elements
in the preceding sentence. In another aspect, a viral vector genome construct
disclosed herein
does not comprise a promoter, a 5' long terminal repeat, a 3' long terminal
repeat, a packaging
signal, a central polypurine tract, or a polyadenylation sequence. In another
aspect, a viral
.. vector genome construct disclosed herein can be used to produce a viral
like particle. In a
further aspect, a long terminal repeat is a self-inactivating long terminal
repeat.
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[057] A viral vector genome construct of the disclosure disclosed herein
may be in the
form of a concatemer. In an aspect, a concatemer may contain one or more
transcription
factors. In a further aspect, a transcription factor may be a ligand-
responsive transcription
factor. In a further aspect, a concatemer may contain one or more antibiotic
selection genes.
Antibiotic selection genes are known in the art. In an aspect, a concatemer is
made and used
as described in Throm et at. Blood, 2009;113(21): 5104-10. For example, a
stable viral
producer cell line can contain fully SIN lentiviral genome and viral accessory
constructs stably
integrated into the genome by concatemeric array transfection. Such array can
be obtained
through the ligation of DNA fragments encoding the SIN lentiviral vector
genome, with drug
resistance and/or other selection/reporter cassettes included into the array.
Viral accessory genes/proteins/constructs
[058] In an aspect, a viral accessory construct encodes one or more
accessory proteins
including for example, structural proteins (e.g., the Gag precursor),
processing proteins (e.g.,
the Pol precursor), and other proteins such as proteases, envelope protein. In
another aspect, a
viral accessary vector comprises sequences that provide the expression and
regulatory signals
needed to manufacture one or more accessory proteins in host cells and
assemble functional
viral particles. In one aspect, coding sequences for an Env, a Rev, and a Gag-
Pol precursor are
on the same plasmid or viral accessory construct. In another aspect, coding
sequences for an
Env, a Rev, and a Gag-Pol precursor are placed on separate plasmids or viral
accessory
constructs. In a further aspect, separate plasmids or viral accessory
constructs are used for each
coding sequence of the Gag, Pol, Rev, and Envelope proteins. In an aspect, a
viral accessory
construct may encode one or more structural and/or regulatory viral proteins,
or functional
fragments or domains thereof, selected from the group consisting of Group-
specific antigen
(Gag), RNA-dependent DNA polymerase (Pol), Regulator of expression of viral
protein (Rev),
Envelope (Env), Transactivator (Tat), Negative regulatory factor (Net), Viral
protein R (Vpr),
Virus infectivity factor (Vif), Viral protein U (Vpu), and Viral protein X
(Vpx). In another
aspect, a functional fragment or domain can comprise one or more proteins
selected from the
group consisting of MA (Matrix [p17]), CA (Capsid [p24]), NC (Nucleocapsid
[p9]), p6,
Protease (p10), RT (p50), RNase H (p15), and Integrase (p31). In an aspect,
coding sequences
of one or more viral accessory proteins are operably linked. In an aspect,
coding sequences of
one or more viral accessory proteins are present on separate viral accessory
constructs.
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[059] In an aspect, a viral accessory construct used here is for producing
a recombinant
lentiviral vector. In an aspect, a viral accessory construct used in the
present disclosure can
comprise one or more of the following elements, separately or collectively, in
any suitable
order or position, e.g., a) a heterologous promoter operably linked to a
polynucleotide sequence
coding for lentivirus Gag and Pol (e.g., a lentivirus Gag-Pol precursor); and
b) a heterologous
promoter operably linked to an env coding sequence.
[060] Any suitable lentiviral 5' LTR can be utilized in accordance with the
present
disclosure, including an LTR obtained from any lentivirus species, sub-
species, strain or clade.
This includes primate and non-primate lentiviruses. Specific examples of
species include, but
are not limited to, e.g., human immunodeficiency virus (HIV)-I (including
subspecies, clades,
or strains, such as A, B, C, D, E, F, and G, R5 and R5X4 viruses, etc.), HIV-2
(including
subspecies, clades, or strains, such as, R5 and R5X4 viruses, etc.), simian
immunodeficiency
virus (Sly), simian-human immunodeficiency virus (SHIV), feline
immunodeficiency virus
(Hy), bovine immunodeficiency virus (BIV), caprine-arthritis-encephalitis
virus, Jembrana
disease virus, ovine lentivirus, visna/maedi virus, and equine infectious
anemia virus.
[061] Genomic reference sequences for such viruses are widely available,
e.g., HIV-I
(NC 001802), HIV-2 (NC 001722), SIV (NC 001549), SIV-2 (NC 004455), caprine
arthritis-encephalitis virus (NC 001463), feline immunodeficiency virus (NC
001482),
Jembrana disease virus (NC 001654), ovine lentivirus (NC 001511), visna/maedi
virus
(NC 001452), equine infectious anemia virus (NC 001450), and bovine
immunodeficiency
virus (NC 001413).
[062] In an aspect, a lentiviral 5' LTR used here comprises signals
utilized in gene
expression, including enhancer, promoter, transcription initiation (capping),
transcription
terminator, and polyadenylation. They are typically described as having U3, R,
and U5 regions.
.. The U3 region of the LTR contains enhancer, promoter and transcriptional
regulatory signals,
including RBEIII, NF-kB, SpI, AP-I and/or GABP motifs. The TATA box is located
about 25
base pairs from the beginning of the R sequence, depending on the species and
strain from
which the 5' LTR was obtained. A completely intact 5' LTR can be utilized, or
a modified copy
can be utilized. Modifications preferably involve the R region, where a TAR
sequence is
substituted (see below), and/or deletion of all or part of a U5 region. The
modified 5' LTR
preferably comprises promoter and enhancer activity, e.g., preferably native
U3, modified R
with a substituted TAR, and native U5.
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[063] In an aspect, a heterologous or non-viral promoter can be operably
linked to a
polynucleotide sequence coding for lentivirus Gag and Pol. By the term
"operably linked," it
is meant that a promoter is positioned in such a way that it can drive
transcription of the recited
coding sequences. In an aspect, gag and pot coding sequences are organized as
the gag-pot
precursor in native lentivirus. The gag sequence codes for a 55-kD Gag
precursor protein, also
called p55. The p55 is cleaved by the virally encoded Protease 4 (a product of
the pot gene)
during the process of maturation into four smaller proteins designated MA
(matrix [p17]), CA
(capsid [p24]), NC (nucleocapsid [p9]), and p6. The Pol precursor protein is
cleaved from Gag
by a virally encoded protease, and further digested to separate the Protease
(p10), RT (p50),
RNase H (p15), and Integrase (p31) activities.
[064] In an aspect, one or more splice donor (SD) sites can be present in a
viral vector
genome construct or a viral accessory construct. A splice donor site is
typically present
between the 3' end of the 5'LTR and the packaging sequence. A downstream
splice acceptor
(SA) can also be present, e.g., at the 3' end of the pot sequences. The SD
site can be present in
multiple copies at any effective locations in the vector. The SD can have a
native or mutated
copy of a lentiviral sequence.
[065] Native gag-pot sequences can be utilized in a viral accessory
construct, or
modifications can be made. These modifications can include, chimeric gag-pot,
where the gag
and pot sequences are obtained from different viruses (e.g., different
species, subspecies,
strains, clades, etc.), and/or where the sequences have been modified to
improve transcription
and/or translation, and/or reduce recombination. In other aspects of the
present disclosure, the
sequences coding for the Gag and Pol precursors (or parts thereof, e.g., one
or more of MA
(matrix [p17]), CA (capsid [p24]), NC (nucleocapsid [p9]), p6, protease (p10),
RT (p50),
RNase H (p15), and integrase (p31)) can be separated and placed on different
vector constructs,
where each sequence has its own expression signals.
[066] The RNA genome of HIV-I contains an approximately 120 nucleotide psi-
packaging signal that is recognized by the nucleocapsid (NC) domain of the Gag
polyprotein
during virus assembly. The critical portions of the packaging signal are
between the major
splice donor (SD) site and the gag initiation codon of the HIV provirus, about
distal to the U5
region of the 5' LTR. In an aspect, a packaging signal is functionally absent
from the accessory
construct to avoid packaging of functionally active gag-pol precursor into the
viral transduction
vector. See, e.g.,U U.S. Pat. No. 5,981,276 (Sodroski et al.), which describes
vectors containing
gag, but which lack the packaging signal.
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[067] Additional promoter and enhancer sequences can be placed upstream of
the 5' LTR
in order to increase, improve, enhance, etc., transcription of the gag-pot
precursor. Examples
of useful promoters, include, mammalian promoters (e.g., constitutive,
inducible, tissue-
specific), CMV, RSV, LTR from other lentiviral species, and other promoters as
mentioned
above and below. In addition, the construct can further comprise transcription
termination
signals, such as a polyA signal that is effective to terminate transcription
driven by the promoter
sequence. Any suitable polyA sequence can be utilized, e.g., sequences from
beta globin
(mammalian, human, rabbit, etc.), thymidine kinase, growth hormone, SV40, and
many others.
[068] In an aspect, gag-pot sequences are placed in opposite
transcriptional orientations
from the envelope sequences in a single viral accessory vector. By the latter,
it is meant that
the direction of transcription is opposite or reversed. This can be achieved
by placing the
corresponding promoters in opposite directions (i.e., facing each other) or
using bi-directional
promoters (e.g., Trinklein et al., Genome Research 14:62- 66, 2004). This
arrangement can be
utilized for safety purposes, e.g., to reduce the risk of recombination and/or
the production of
functional recombinant HIV genomes. Safety is increased with such vectors as
there is no
possibility that transcriptional read-through would result in a RNA that
contains both functional
gag-pot and env sequences. Transcriptional interference can be prevented by
utilizing strong
polyadenylation sequences that terminate transcription. Examples of strong
transcription
termination sequences are known in the art, including, e.g., rabbit beta-
globin polyadenylation
signal (Lanoix and Acheson, EMBO J. 1988 Aug;7(8):2515-22), See, also Plant et
at.,
Molecular and Cellular Biology, April 2005, 25(8): 3276-3285. In addition,
other elements
can be inserted between the gag-pot and env coding sequences to facilitate
transcriptional
termination, such as a cis-acting ribozyme, or an RNAi sequence which are
targeted to any
putative read-through sequence. Similarly, instability sequences, termination
sequences, and
pause sites can be placed between the coding sequences.
[069] In an aspect, a viral accessory construct may encode structural viral
proteins. In an
aspect, a viral accessory construct may encode regulatory viral proteins. In
an aspect, a viral
accessory construct may encode both structural and regulatory viral proteins.
[070] In an aspect, a viral accessory construct may encode structural
and/or regulatory
viral proteins that include, but are not limited to Group-specific antigen
(Gag), DNA
polymerase (Pol), Regulator of expression of viral protein (Rev), Envelope
(Env),
Transactivator (Tat), Negative regulatory factor (Nef), Viral protein R (Vpr),
Virus infectivity
factor (Vif), Viral protein U (Vpu), and Viral protein X (Vpx).
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[071] Gag encodes structural proteins such as Matrix protein (MA),
Capsid protein (CA),
and Nucleocapsid protein (NC). Pot encodes proteins such as Protease (PR),
Reverse
transcriptase (RT), and Integrase (IN). Env encodes surface and transmembrane
units of
envelope protein.
[072] In an aspect, encoded viral accessory proteins are fusion proteins.
In an aspect,
encoded viral accessory proteins are partial viral accessory proteins, such as
protein domains.
In an aspect, viral accessory protein domains may include, but are not limited
to capsid protein
(CA), matrix protein (MA), nucleocapsid protein (NC), p6, transcription factor
specificity
protein 1 (SP1), reverse transcriptase (RT), integrase (IN), protease (PR),
and deoxyuridine
triphosphatase (dUTPase or DU). In a further aspect, encoded viral accessory
proteins include
at least one full length protein or at least one protein domain.
[073] In an aspect, a viral construct can further comprise an RRE
element, including an
RRE element which is obtained from a different lentiviral species than the 5'
LTR or gag and
poi sequences. The RRE element is the binding site for the rev polypeptide
which is a 13-kD
sequence-specific RNA binding protein. Constructs which contain the RRE
sequence depend
on the Rev polypeptide for efficient expression. Rev binds to a 240-base
region of complex
RNA secondary structure of the Rev response element ("RRE") that is located
within the
second intron of HIV, distal to the pot and gag coding sequences. The binding
of Rev to RRE
facilitates the export of unspliced and incompletely spliced viral RNAs from
the nucleus to the
cytoplasm, thereby regulating the expression of HIV proteins. The RRE element
can be in any
suitable position on the construct, preferably following the Gag-Pol precursor
in its
approximate native position. Similarly for the Tat polypeptide, any suitable
Rev polypeptide
can be utilized as long as it retains the ability to bind to RRE.
Viral capsids/envelopes
[074] Virus particles contain a viral genome packaged in a protein coat
called the capsid.
For some viruses, the capsid is surrounded by lipid bilayer that contains
viral proteins, usually
including the proteins that enable the virus to bind to the host cells. This
lipid and protein
structure is called the virus envelope, and is derived from the host cell
membranes. The capsid
and envelope play many roles in viral infection, including virus attachment to
cells, entry into
cells, release of the capsid contents into the cells, and packaging of newly
formed viral
particles. The capsid and envelope are also responsible for transfer of the
viral genetic material
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from one cell to another. These structures also determine the stability
characteristics of the
virus particle, such as resistance to chemical or physical inactivation.
[075] In an aspect, a stable viral vector producer cell line produces an
envelope protein.
In an aspect, envelope protein(s) employed in this cell line system use either
the native HIV
env gene (wild-type or codon optimized) or generate a pseudotyped particle
using a
biocompatible substitute including, but not limited to, amphotropic envelope
protein, vesicular
stomatitis vector (Indiana or other strain), measles or bioengineered chimeric
measles envelope
proteins, gibbon ape leukemia virus, or feline leukemia virus or bioengineered
FLV chimeras.
[076] In an aspect, viral vectors disclosed herein contain one or more
capsid proteins. In
an aspect, capsid proteins may be heterologous. In an aspect, capsid proteins
may be
genetically modified. In a further aspect, capsid proteins may be chemically
modified.
Strategies to genetically and chemically modify capsid proteins are known in
the art. Capsid
proteins may be modified in order to alter vector biodistribution.
[077] In an aspect, viral vectors disclosed herein may have sequences
encoding for one or
more envelope ("Env") proteins. Viral vector tropism is determined by the
ability of the viral
envelope protein to interact with molecules (proteins, lipids, or sugars) on
the host cell.
[078] In an aspect, a viral accessory construct can comprise an envelope
module or
expression cassette comprising a heterologous promoter operably linked to an
env coding
sequence. The envelope polypeptide is displayed on the viral surface and is
involved in the
recognition and infection of host cells by a virus particle. The host range
and specificity can be
changed by modifying or substituting the envelope polypeptide, e.g., with an
envelope
expressed by a different (heterologous) viral species or which has otherwise
been modified.
This is called pseudotyping. See, e.g., Yee et al., Proc. Natl. Acad. Sci. USA
91: 9564-9568,
1994. Vesicular stomatitis virus (VSV) protein G (VSV G) has been used
extensively because
of its broad species and tissue tropism and its ability to confer physical
stability and high
infectivity to vector particles. See, e.g., Yee et at., Methods Cell Biol.,
(1994) 43:99-112.
[079] An envelope polypeptide can be utilized without limitation,
including, e.g., HIV
gp120 (including native and modified forms), Moloney murine leukemia virus
(MoMuLV or
MMLV), Harvey murine sarcoma virus (HaMuSV or HSV), murine mammary tumor virus
(MuMTV or MMTV), gibbon ape leukemia virus (GALV), Rous sarcoma virus (RSV),
hepatitis viruses, influenza viruses (VSV-G), Mokola virus, rabies, filovirus
(e.g., Ebola and
Marburg, such as GP1/GP2 envelope, including NP 066246 and Q05320),
amphotropic,
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alphavirus, etc. Other examples include, e.g., envelope proteins from
Togaviridae,
Rhabdoviridae, Retroviridae, Poxviridae, Paramyxoviridae, and other enveloped
virus
families. Other example envelopes are from viruses listed in the following
database located on
the worldwide web at ncbi.nlm.nih.gov/genome/viruses.
[080] Furthermore, a viral envelope protein can be modified or engineered
to contain
polypeptide sequences that allow the transduction vector to target and infect
host cells outside
its normal range or more specifically limit transduction to a cell or tissue
type. For example,
the envelope protein can be joined in-frame with targeting sequences, such as
receptor ligands,
antibodies (using an antigen-binding portion of an antibody or a recombinant
antibody-type
.. molecule, such as a single chain antibody), and polypeptide moieties or
modifications thereof
(e.g., where a glycosylation site is present in the targeting sequence) that,
when displayed on
the transduction vector coat, facilitate directed delivery of the virion
particle to a target cell of
interest. Furthermore, envelope proteins can further comprise sequences that
modulate cell
function. Modulating cell function with a transducing vector may increase or
decrease
transduction efficiency for certain cell types in a mixed population of cells.
For example, stem
cells could be transduced more specifically with envelope sequences containing
ligands or
binding partners that bind specifically to stem cells, rather than other cell
types that are found
in the blood or bone marrow. Such ligands are known in the art. Non-limiting
examples are
stem cell factor (SCF) and Flt-3 ligand. Other examples, include, e.g.,
antibodies (e.g., single-
chain antibodies that are specific for a cell-type), and essentially any
antigen (including
receptors) that is specific for such tissues as lung, liver, pancreas, heart,
endothelial, smooth,
breast, prostate, epithelial, etc.
[081] Any heterologous promoter can be utilized to drive expression of the
viral envelope
coding sequence when operably linked to it. Examples include, e.g., CMV, EF 1
alpha, EF 1
alpha-HTLV-1 hybrid promoter, ferritin promoters, inducible promoters,
constitutive
promoters, and other promoters mentioned herein.
[082] In an aspect, encoded envelope proteins are endogenous. In a further
aspect,
encoded envelope proteins are heterologous. Heterologous envelope proteins of
the viral
vectors disclosed herein may be generated using any envelope protein that is
biocompatible.
Biocompatibility can be determined using methods known in the art.
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[083] In an aspect, env may be derived from human immunodeficiency virus
(HIV). In
an aspect, a sequence encoding an HIV-derived envelope gene may be wild-type.
In a further
aspect, a sequence encoding an HIV-derived envelope gene may be codon-
optimized.
[084] Env may also be generated as a pseudotyped particle. Pseudotyping
enables the
engineering of viral vector particles with different target cell
specificities, to expand and/or to
alter the host range of the native virus from which the envelope protein was
derived.
[085] In an aspect, the viral vectors disclosed herein may be amphotropic
pseudotyped
viral vectors. In an aspect, the viral vectors disclosed herein may be
ecotropic pseudotyped
viral vectors. In an aspect, the viral vectors disclosed herein may be
pantropic pseudotyped
viral vectors. Envelope protein sequences encoded by the viral vectors
disclosed herein may
be derived from any species of the genera Vesiculovirus, Gammaretrovirus, or
Morbillivirus.
[086] In an aspect, envelope proteins may be derived from a species of the
Vesiculovirus
genus including, but not limited to, vesicular stomatitis New Jersey virus
(VSV-NJ), and
vesicular stomatitis Indiana virus (VSV-IN). In a further aspect, envelope
proteins may be
derived from any vesicular stomatitis virus serotype. In a further aspect,
envelope proteins
may be truncated proteins. In a further aspect, envelope proteins may be
bioengineered
chimeric vesiculovirus proteins.
[087] In an aspect, envelope proteins may be derived from a species of the
Gammaretrovirus genus, including, but not limited to gibbon ape leukemia virus
(GaLV) and
feline leukemia virus (FLV). In a further aspect, envelope proteins may be
bioengineered
chimeric gammaretrovirus proteins, including GaLV chimeras and FLV chimeras. A
"chimera" as defined herein refers to a biological entity, such as a virus,
that is composed of
two or more genetic fragments of distinct origin or of distinct composition.
[088] In an aspect, envelope proteins may be derived from a species of the
Morbillivirus
genus including, but not limited to, measles virus. In a further aspect,
envelope proteins may
be bioengineered chimeric morbillivirus proteins, including bioengineered
chimeric measles
envelope proteins. Methods of bioengineering chimeric envelope proteins are
known in the
art.
Optional Tat
[089] In an aspect, a stable viral vector producer cell line comprises or
produces a Tat
protein. In another aspect, a stable viral vector producer cell line does not
produce a Tat
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protein. In the absence of a Tat protein, a lentiviral genome vector is
modified such that the
HIV promoter in the 5' LTR is replaced with a heterologous enhancer/promoter
to ensure
transcription. In an aspect, such promoter could be either viral (like CMV) or
cellular (like
EF1-a).
[090] In another aspect, a viral accessory construct can further comprise a
TAR element
that is obtained from a different lentiviral species, group, sub-species, sub-
group, strain, or
clade than the 5' LTR and/or the gag and poi sequences that are present in it,
i.e., it is
heterologous to other lentiviral elements present in the construct. The TAR is
preferably
present in the 5' LTR in its normal location, e.g., between the U3 and U5
elements of the LTR,
e.g., where the native R is replaced by R' of a heterologous lentiviral
species.
[091] The TAR element is a trans-activating response region or response
element that is
located in the 5'LTR (e.g., R) of the viral DNA and at the 5' terminus of the
corresponding
RNA. When present in the lentiviral RNA, the transcriptional transactivator,
Tat, binds to it,
activating transcription from the HIV LTR many-fold. Tat is an RNA binding
protein that
binds to a short-stem loop structure formed by the TAR element.
[092] When a heterologous TAR element is utilized, the 5' LTR can be
modified routinely
by substituting its native TAR for a TAR sequence from another species.
Examples of TAR
regions are widely known. See, e.g., De Areliano et at., AIDS Res. Human
Retro., 2005, 21:
949-954. Such a modified lentiviral 5' LTR can comprise intact U3 and U5
regions, such that
the LTR is completely functional. The TAR region or the entire R can be
substituted.
[093] As indicated above, the Tat polypeptide binds to the TAR sequence.
The coding
sequence for tat can be present in a viral accessory construct. Any Tat
polypeptide can be
utilized as long as it is capable of binding to TAR and activating
transcription of the RNA.
This includes native tat sequences which are obtained from the same or
different species as the
.. cognate TAR element, as well as engineered and modified tat sequences.
Promoters
[094] In an aspect, a construct disclosed here contains one or more
expression cassettes
that express an accessory protein or RNA molecule under the control of a
constitutive,
inducible, switched, recombined, disrupted/edited promoter or
promoter/enhancer. In an
aspect, a promoter is a minimal promoter with upstream cis regulatory to
determine spatio-
temporal expression pattern of the promoter. Upstream regulatory elements may
include cis-
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acting elements (or cis-acting motifs) or transcription factor binding sites.
In a further aspect,
the promoter comprises a combination of heterologous upstream regulatory
elements.
[095] In an aspect, a promoter is a promoter/enhancer. As used herein,
the term
promoter/enhancer refers to a segment of DNA that contains sequences capable
of providing
both promoter and enhancer functions. The promoter/enhancer may be endogenous
or
exogenous or heterologous. An endogenous promoter/enhancer is one which is
naturally linked
with a given gene in a native viral genome. An exogenous or heterologous
enhancer/promoter
is one which is placed in juxtaposition to a gene by means of molecular
biology techniques
such that the transcription of that gene is directed by the linked
promoter/enhancer.
[096] In an aspect, a promoter is an inducible promoter. In an aspect, an
inducible
promoter is positively inducible and regulated by positive control. In an
aspect, an inducible
promoter is negatively inducible, and regulated by negative control.
[097] In a further aspect, an inducible promoter may be a chemically
inducible promoter.
Chemically inducible promoters are known in the art. In a further aspect, a
chemically
inducible promoter may be a tetracycline-controllable promoter. In a further
aspect, a
tetracycline-controllable promoter is a natural promoter. In a further aspect,
a tetracycline-
controllable promoter is a synthetic promoter.
[098] In a further aspect, an inducible promoter may be a temperature
inducible promoter.
In a further aspect, an inducible promoter may be a light inducible promoter.
In a further aspect,
an inducible promoter may be a physiologically regulated promoter.
[099] In an aspect, a promoter may be a constitutive promoter. In an
aspect, a promoter
may be a switched promoter. In an aspect, a promoter may be a recombined
promoter. In an
aspect, a promoter may be a disrupted/edited promoter.
[0100] In an aspect, a promoter element may be naturally derivable. In a
further aspect, a
promoter may contain sequences derived from a eukaryotic promoter including,
but not limited
to CMV, EFla, SV40, PGK1, Ubc, human beta actin, CAG, TRE, CaMKIIa, Call, 10,
H1, and
U6.
[0101] In a further aspect, a promoter comprises synthetic elements.
Methods to prepare
synthetic promoters are known in the art. In an aspect, a synthetic promoter
is a constitutive
synthetic promoter. In an aspect, a synthetic promoter is an inducible
synthetic promoter. In
an aspect, a synthetic promoter is a tissue-specific synthetic promoter.
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Polyadenylation sequences
[0102] In an aspect, a viral vector genome construct or a viral
accessory construct
comprises one or more polyadenylation sequences (p(A)). Expression of
recombinant DNA
sequences in eukaryotic cells requires expression of signals to direct
termination and
.. polyadenylation of the resulting transcript. The term "polyadenylation
sequence" as used
herein refers to a nucleic acid sequence that directs the termination and
polyadenylation of a
nascent formed RNA transcript. Transcripts lacking a polyA tail may be
unstable and quickly
degraded. A polyA signal utilized in a viral vector genome construct disclosed
herein may be
heterologous or endogenous. An endogenous polyA signal refers to a polyA
sequence that is
found naturally at the 3' end of the coding region of a given gene. A
heterologous polyA signal
refers to a polyA sequence that is isolated from one gene and placed at the 3'
end of another
gene.
Expression cassettes
[0103] In an aspect, a viral vector genome construct and/or a viral
accessory construct
described here comprise one or more expression cassettes. Expression cassettes
may be a
monocistronic expression cassette or a polycistronic expression cassette.
[0104] In an aspect, a polycistronic expression cassette contains one or
more viral skip
sequences. Viral skip sequences are "self-cleaving" 2A peptides, which are 18-
22 amino acid
viral oligopeptides that mediate "cleavage" of polypeptides during translation
in eukaryotic
.. cells. The "2A" designation refers to a specific region of the viral
genome. The mechanism
of 2A cleavage is ribosome skipping, mediated by a highly conserved C-terminal
sequences
essential to the creation of steric hindrance. In an aspect, viral skip
sequences may include 2A
peptides derived from porcine teschovirus-1 2A (P2A). In an aspect, viral skip
sequences may
include 2A peptides derived from Thosea asigna virus 2A (T2A). In an aspect,
viral skip
sequences may include 2A peptides derived from equine rhinitis A virus (E2A).
In an aspect,
viral skip sequences may include 2A peptides derived from foot-and-mouth
disease virus
(F2A). In a further aspect, viral skip sequences may be derived from any virus
with a 2A
sequence substantially similar to the conserved "2A" C-terminal sequence
GDVEXNPGP.
[0105] In an aspect, a polycistronic expression cassette contains one or
more internal
ribosome entry site elements (IRES). An IRES element is a cis-acting RNA
region that
promotes internal initiation of protein synthesis. An IRES sequence is
recognized by a
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ribosome, and can therefore be used to drive translation of multiple proteins
off a single
transcript.
[0106] In a further aspect, a polycistronic expression cassette contains
one or more viral
skip sequences and one or more internal ribosome entry site elements.
[0107] In an aspect, a polycistronic expression cassette encodes for
sequences that provide
a similar mechanism to viral skip sequences or internal ribosome entry
sequences.
Codon optimization
[0108] Expression cassettes contain sequences that encode one or more
viral accessory
proteins. In an aspect, a viral accessory protein may be encoded by a wild-
type sequence. In
an aspect, a viral accessory protein may be encoded by a mutated sequence. In
a further aspect,
a viral Integrase is encoded by a mutated sequence. In a further aspect, a
viral accessory protein
may be encoded by a codon optimized sequence. Codon optimization is commonly
used to
increase production of recombinant proteins or viral vectors. Codon
optimization is a desirable
molecular tool to address codon usage bias. Codon usage bias is a feature of
all genomes, and
.. reflects the frequency of codon distribution within a genome is referred to
as codon usage bias.
Codon usage is variable between species, and preferred codons are more
frequently used in
highly expressed genes. Transfer RNAs, or tRNAs, reflect the codon usage in a
given organism,
and therefore the abundance of particular tRNAs is variable between organisms.
Codon
optimization is a process by which DNA sequences are modified by introducing
silent
mutations to generate synonymous codons.
[0109] In a further aspect, an expression cassette may contain sequences
that are all wild-
type sequences, all codon optimized sequences, all mutated sequences, or a
combination of
wild type, codon optimized, and mutated sequences. In an aspect, expression of
Rev, Tat, Nef,
Vpr, Vif, Vpu/Vpx when included, is from wild-type or codon optimized
constructs which are
polycistronic using viral skip sequences (such as P2A, or T2A) or internal
ribosome entry
sequences or other similar mechanism or as a single message per transcript. In
an aspect,
expression of gag/pol is from a wild-type or codon optimized polycistronic
message, or as
separate gag and pol constructs, or as further separated CA, MA SP1, NC, p6,
RT, IN, PR,
and/or DU constructs.
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Introducing viral vectors to target or host cells
[0110] In an aspect, the introduction of one or more constructs into a
cell is achieved using
a standard chemical, biological, or physical methods including, but not
limited to,
lipofectamine or lipofectamine-like chemical reagents, polyethyleneimine
(PEI), calcium
phosphate crystals, retroviral vector, lentiviral vector, nanoparticles or
nanoparticle-like
reagents, or electroporation. In another aspect, incorporation of these
constructs into the cell
line genome is achieved using biological recombinant enzymes including, but
not limited to,
integrase, transposase, recombinase, the CRISPR-Cas9 system, or utilizing
spontaneous or
targeted insertion using cellular DNA repair machinery.
[0111] In an aspect, methods of introducing viral vector constructs to a
target or host cell
may include transduction or transfection. Transfection and transduction may be
performed
using a variety of techniques known in the art, and may include optimizations
for enhancing
transfection or transduction efficiency. In an aspect, optimization may
comprise freeze-
thawing reagents.
is [0112] In an aspect, viral vector constructs are introduced to
target or host cells using
chemical methods known in the art. In an aspect, viral vector constructs are
introduced to target
or host cells using biological methods known in the art. In an aspect, viral
vector constructs
are introduced to target or host cells using physical methods known in the
art.
[0113] In an aspect, viral vector constructs may be introduced to a
target or host cell by
methods comprising optical techniques. In an aspect, viral vector constructs
may be introduced
to a target or host cell by methods comprising magnetic techniques. In an
aspect, viral vector
constructs may be introduced to a target or host cell by methods comprising
biolistic
techniques. In an aspect, viral vector constructs may be introduced to a
target or host cell by
methods comprising polymer-based techniques. In an aspect, viral vector
constructs may be
introduced to a target or host cell by methods comprising liposome-based
techniques. In an
aspect, viral vector constructs may be introduced to a target or host cell by
methods comprising
nanoparticle-based techniques. In a further aspect, viral vector constructs
may be introduced to
a target or host cell by a combination of methods comprising a combination of
techniques
including, but not limited to optical, magnetic, biolistic, polymer-based,
liposome-based, and
nanoparticle-based techniques.
[0114] In a further aspect, viral vector constructs may be introduced to
a target or host cell
by methods comprising electroporation. In a further aspect, viral vector
constructs may be
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introduced to a target or host cell by methods comprising sonoporation. In a
further aspect,
viral vector constructs may be introduced to a target or host cell by methods
comprising
mechanoporation. In a further aspect, viral vector constructs may be
introduced to a target or
host cell by methods comprising photoporation.
[0115] In a further aspect, methods of introduction may also comprise
methods that involve
use of a cationic polymer, calcium phosphate, cationic lipid, or a combination
thereof In an
aspect, a cationic polymer is hexadimethrine bromide (commercial brand name
Polybrene).
[0116] In a further aspect, methods of introduction may also comprise
methods that involve
use of a retrovirus, lentivirus, transposon, transcription activator-like
effector nuclease
(TALEN), Zinc Finger nuclease, meganuclease, transposase, a CRISPR-related
nuclease (e.g.,
Cas9, Cas12a, etc.), or recombinase. In an aspect, a recombinase may be a Cre-
recombinase,
Flippase recombinase, or a derivative thereof
[0117] Methods to promote the integration of nucleic acids into
production cells are known
in the art, and can include, but are not limited to, linearizing a nucleic
acid construct.
[0118] In an aspect, one or more viral vector constructs may be stably
integrated or
episomally maintained within the viral vector production cell. Gene expression
of sequences
encoded by any of the introduced viral vectors may occur from integrated
sequences or
epi some s.
[0119] In an aspect, a viral vector production cell stably expressing
some of the
components may be transfected with remaining components that are required for
vector
production. Transfection of the remaining components required for viral vector
production
may be transient.
[0120] A viral vector construct may integrate randomly or in a site-
specific manner upon
introduction into a host or target cell.
.. Viral vector production cells
[0121] The disclosure disclosed herein provides a method of making viral
vector particles
in vitro by introducing one or more viral vector constructs of the disclosure
into a compatible
target cell or host cell and growing the cell under conditions which result in
cell expansion and
expression of the vector components. The terms "target cell" and "host cell"
as used herein are
interchangeable.
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[0122] A viral vector production cell is a target cell or host cell that
is capable of producing
a viral vector or viral vector particle upon introduction of one or more viral
vector constructs.
[0123] In an aspect, a viral vector production cell is a transgenic
cell. As used herein, the
term "transgenic cell" refers to a cell comprising genetic material that has
been transferred from
one cell type to another cell type. In an aspect, a viral vector production
cell population is
polyclonal. Polyclonal cells comprise a heterogeneous population of cells with
multiple clones
that may have variations in the number of integration events and sites of
integration across the
cells. In a further aspect, a viral vector production cell population is
monoclonal.
[0124] In an aspect, a viral vector production cell is from a cell line
that has been expanded
from a selected viral vector production cell clone.
[0125] Viral vector production cell clones may be derived from a
polyclonal population by
methods known in the art. Methods of selection include, but are not limited
to, limiting
dilution, single cell sorting, single cell selection, and combinations
thereof. Limiting dilution
may be performed by methods known in the art. Single cell sorting may be
performed by
methods known in the art, including, but not limited to, single cell printing,
fluorescence
activated cell sorting (FACS), and magnetic activated cell sorting. Single
cell selection may
be performed by selection methods known in the art, including, but not limited
to selection for
an epitope, a protein, a reporter gene, or combination thereof. In a further
aspect, single cell
selection methods may comprise selection via one or more metabolic or
antibiotic properties.
[0126] In an aspect, viral vector production cell clones or cell lines grow
in an adherent
manner. In an aspect, viral vector production cell clones or cell lines grow
in suspension. In a
further aspect, adherent viral vector production cell clones or cell lines may
be suspension-
adapted.
[0127] In an aspect, viral vector production cell clones or cell lines
are cultured in serum-
supplemented or serum-free media. A person of skill in the art will be able to
select an
appropriate media for the given viral vector production cell type, and to
modify the media
composition at various stages of the method disclosed herein. Media may have a
selection of
secreted cellular proteins, diffusible nutrients, amino acids, organic salts,
inorganic salts,
vitamins, trace metals, sugars, and other growth-promoting substances such as
cytokines.
Media may be supplemented with glutamine or an alternative thereof
[0128] In an aspect, viral vector production cell clones or cell lines
may be any eukaryotic
cell that supports the lifecycle of the specific virus from which the vector
is derived. In an
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aspect, for a retroviral vector, a production cell clones or cell lines may be
any eukaryotic cell
that supports a retrovirus life cycle. In an aspect, for a lentiviral vector,
a production cell clones
or cell lines may be any eukaryotic cell that supports a lentivirus life
cycle. In an aspect, for a
herpesvirus vector, a production cell clones or cell lines may be any
eukaryotic cell that
supports a herpesvirus life cycle. In an aspect, for an adenoviral vector, a
production cell clones
or cell lines may be any eukaryotic cell that supports an adenovirus life
cycle. In an aspect, for
an adeno-associated viral vector, a production cell clones or cell lines may
be any eukaryotic
cell that supports an adeno-associated virus life cycle.
[0129] In an aspect, viral vector production cell clones or cell lines
are immortalized. Cell
lines may be commercially available or non-commercially available laboratory-
derivatives. In
a further aspect, viral vector production cell clones or cell lines are of
eukaryotic origin. In an
aspect, viral vector production cell clones or cell lines are of mammalian
origin. Mammalian
cells for the production of viral vectors are known in the art. In an aspect,
viral vector
production cell clones or cell lines are of human origin.
[0130] In a further aspect, a viral vector producer cell line is developed
in or from Human
Embryonic Kidney (HEK) 293 cells, which are highly transfectable. In a further
aspect, a viral
vector producer cell line is a derivate of HEK293 cells, such as HEK293T or
HEK293F cells.
In a further aspect, cell types for viral vector production cell clones or
cell lines include, but
are not limited to, HeLa cells, Vero cells, Chinese Hamster Ovary (CHO) cells,
A549 cells,
.. and NIH 3T3 cells.
Characterization of produced viral vector
[0131] Viral vector particles produced by a viral vector producer cell
clone or cell line may
be characterized by a variety of methods known to those of skill in the art.
[0132] In an aspect, a viral vector particle produced by a method
disclosed herein is a
psuedotyped viral particle. Pseudotyped viral particles may be produced by
substituting viral
attachment proteins from one viral serotype with another. As used herein, a
"viral attachment
protein" refers to a viral capsid protein or a viral envelope protein.
[0133] In an aspect, a viral vector particle produced by a method
disclosed herein is a
mosaic viral particle. Mosaic viral particles may be produced by mixing
different viral
attachment proteins from different viral variants.
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[0134] In an aspect, a viral vector particle produced by a method
disclosed herein is a
chimeric viral particle. Chimeric viral particles may be produced by methods
that include
swapping smaller domains of viral attachment proteins between serotypes (via
rational methods
or high throughput recombination techniques).
[0135] From a stable viral vector producing cell clone or cell line, viral
vector genome and
accessory proteins may be characterized quantitatively or qualitatively. In an
aspect, the
stoichiometric ratio of viral vector genome and one or more accessory proteins
may be
determined. In a further aspect, the level of viral vector genome and one or
more accessory
proteins may be determined.
[0136] An integration profile of a selected cell clone or cell line may be
determined. In an
aspect, an integration profile or an insertional profile may be detected by
methods known in
the art such as inverse PCR, linear amplification-mediated PCR or ligation-
mediated PCR.
Vector flanking sequences detected by such methods can then be mapped to a
host cell genome
and compared to a reference set. Mapping can be performed using computational
tools to map
and analyze vector-flanking sequences, such as QuickMap.
[0137] In an aspect, recombinant viral vectors may be harvested from a
cell clone or a cell
line. In an aspect, the cell line is monoclonal. Harvested viral vectors may
be characterized
qualitatively or quantitatively. In an aspect, viral titer is expressed in
transducing units per
milliliter (t.u./m1).
[0138] Viral titer may be determined using physical or functional
titration. In an aspect,
titration methods include but are not limited to transduction of indicator
cells using dose-
dependent quantities of vector supernatant.
[0139] In a further aspect, transduced indicator cells may be assessed
using polymerase
chain reaction (PCR). Quantification by PCR may be performed using relative
quantification
or absolute quantification. Methods for relative or absolute quantification by
PCR are known
in the art.
[0140] In a further aspect, methods of viral titer determination are
enzyme immunoassays.
Harvested viral particles may be quantified by measuring the amount of a viral
capsid protein
using immunoassays specific to the virus from which the viral capsid protein
was derived (for
example, p24 for HIV).
[0141] Viral vector particles produced by methods disclosed herein may
be concentrated
and/or purified using flow-through ultracentrifugation and high-speed
centrifugation, and
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tangential flow filtration. Flow through ultracentrifugation has been used for
the purification
of RNA tumor viruses (Toplin et at., Applied Microbiology, 1967, 15: 582-589;
Burger et at.,
Journal of the National Cancer Institute, 1970, 45: 499-503). The present
disclosure provides
the use of flow-through ultracentrifugation for the purification of lentiviral
vectors. This
method can comprise one or more of the following steps. For example, a
lentiviral vector can
be produced from cells using a cell factory or bioreactor system. A transient
transfection system
(see above) can be used or packaging or producer cell lines can also similarly
be used. A pre-
clarification step prior to loading the material into the ultracentrifuge
could be used if desired.
Flow-through ultracentrifugation can be performed using continuous flow or
batch
sedimentation. The materials used for sedimentation are, e.g.: Cesium chloride
(CsC1),
potassium tartrate and potassium bromide, which create high densities with low
viscosity
although they are all corrosive. CsC1 is frequently used for process
development as a high
degree of purity can be achieved due to the wide density gradient that can be
created (1.0 to
1.9 g/cm ). Potassium bromide can be used at high densities, but only at
elevated temperatures,
i.e. 25 C, which may be incompatible with stability of some proteins. Sucrose
is widely used
due to being inexpensive, non-toxic and can form a gradient suitable for
separation of most
proteins, sub-cellular fractions and whole cells. Typically Hie maximum
density is about 1.3
g/cm3. The osmotic potential of sucrose can be toxic to cells in which case a
complex gradient
material can be used, e.g. Nycodenz. A gradient can be used with 1 or more
steps in the
.. gradient. A preferred aspect is to use a step sucrose gradient. The volume
of material can is
preferably from 0.5 liters to over 200 liters per run. The flow rate speed is
preferably from 5 to
over 25 liters per hour. The preferred operating speed is between 25,000 and
40,500 rpm
producing a force of up to 122,000x g. The rotor can be unloaded statically in
desired volume
fractions. A preferred aspect is to unload the centrifuged material in 100m1
fractions. The
isolated fraction containing the purified and concentrated lentiviral vector
can then be
exchanged in a desired buffer using gel filtration or size exclusion
chromatography. Anionic
or cationic exchange chromatography could also be used as an alternate or
additional method
for buffer exchange or further purification. In addition, Tangential Flow
Filtration can also be
used for buffer exchange and final formulation if required. Tangential Flow
Filtration (TFF)
.. can also be used as an alternative step to ultra or high speed
centrifugation, where a two-step
TFF procedure would be implemented. The first step would reduce the volume of
the vector
supernatant, while the second step would be used for buffer exchange, final
formulation and
some further concentration of the material. The TFF membrane should have a
membrane size
of between 100 and 500 kilodaltons, where the first TFF step should have a
preferable
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membrane size of 500 kilodaltons, while the second TFF should have a
preferable membrane
size of between 300 to 500 kilodaltons. The final buffer should contain
materials that allow the
vector to be stored for long term storage.
[0142] The present disclosure also provides methods for the
concentration and purification
of lentiviral vectors using either cell factories that contains adherent
cells, or a bioreactor that
contains suspension cells that are either transfected or transduced with the
vector and accessory
constructs to produce lentiviral vector. Non-limiting examples or bioreactors,
include the
Wave bioreactor system and the Xcellerex bioreactors. Both are disposable
systems. However
non-disposable systems can also be used. The constructs can be those described
herein, as well
as other recombinant viral vectors. Alternatively the cell line can be
engineered to produce
lentiviral vector without the need for transduction or transfection. After
transfection, the
lentiviral vector can be harvested and filtered to remove particulates and
then is centrifuged
using continuous flow high-speed or ultracentrifugation. In an aspect, a high
speed continuous
flow device like the JCF-A zonal and continuous flow rotor with a high speed
centrifuge is
used. Also provided is any continuous flow centrifuge where the speed of
centrifugation is
greater than 5,000xg RCF and less than 26,000x g RCF. Preferably, the
continuous flow
centrifugal force is about 10,500x g to 23,500 x g RCF with a spin time of
between 20 hours
and 4 hours, with longer centrifugal times being used with slower centrifugal
force. The
lentiviral vector can be centrifuged on a cushion of more dense material (a
non-limiting
example is sucrose but other reagents can be used to form the cushion and
these are well known
in the art) so that the lentiviral vector does not form aggregates that are
not filterable, as is the
problem with straight centrifugation of the vector that results in a viral
vector pellet.
Continuous flow centrifugation onto a cushion allows the vector to avoid large
aggregate
formation, yet allows the vector to be concentrated to high levels from large
volumes of
transfected material that produces the lentiviral vector. In addition, a
second less-dense layer
of sucrose can be used to band the lentiviral vector preparation. The flow
rate for the continuous
flow centrifuge is preferably between 1 and 100m1 per minute, but higher and
lower flow rates
can also be used. The flow rate is adjusted to provide ample time for the
vector to enter the
core of the centrifuge without significant amounts of vector being lost due to
the high flow rate.
If a higher flow rate is desired, then the material flowing out of the
continuous flow centrifuge
can be re-circulated and passed through the centrifuge a second time. After
the virus is
concentrated using continuous flow centrifugation, the vector can be further
concentrated using
Tangential Flow Filtration (TFF), or the TFF system can be simply used for
buffer exchange.
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A non-limiting example of a TFF system is the Xampler cartridge system that is
produced by
GF> Healthcare. Preferred cartridges are those with a MW cut-off of 500,000 MW
or less.
Preferably a cartridge is used with a MW cut-off of 300,000 MW. A cartridge of
100,000MW
cut-off can also be used. For larger volumes, larger cartridges can be used
and it will be easy
for those in the art to find the right TFF system for this final buffer
exchange and/or
concentration step prior to final fill of the vector preparation. The final
fill preparation may
contain factors that stabilize the vector. For example, sugars are generally
used and are known
in the art.
Further cell line modification
[0143] In an aspect, a cell line utilized to manufacture a recombinant
viral vector can be
modified in any of the ways mentioned below to enhance viral vector
production, e.g., by the
introduction of RNAi or antisense to knock-out genes that reduce the
expression of genes that
limit viral vector production, or by the introduction of sequences that
enhance viral vector
production. Sequences that code for cellular or viral enhancers can also be
engineered into cell
lines (e.g., using additional plasmid vectors), such as herpes virus,
hepatitis B virus, which act
on HIV LTRs to enhance the level of virus product, or cellular transactivator
proteins. Cellular
transactivation proteins include, e.g., NF-kB, UV light responsive factors,
and T cell activation
factors. In another aspect, a cell line utilized to manufacture a recombinant
viral vector can be
modified or edited by a nuclease selected from the group consisting of a
meganuclease, a zinc-
finger nuclease (ZFN), a transcription activator-like effector nuclease
(TALEN), a CRISPR-
related nuclease (e.g., Cas9, Cas12a, etc.).
[0144] In an aspect, a cell line can be transformed routinely with
construct DNA, e.g., using
electroporation, calcium phosphate, liposomes, etc., to introduce the DNA into
cells. Cells can
be co-transformed (i.e., using both accessory and transfer vectors), or they
can be transformed
in separate steps, where each step involves the introduction of a different
vector.
[0145] Cells are cultured under conditions effective to produce viral
vectors. Such
conditions include, e.g., the particular milieu needed to achieve protein
production. Such a
milieu, includes, e.g., appropriate buffers, oxidizing agents, reducing
agents, pH, co-factors,
temperature, ion concentrations, suitable age and/or stage of cell (such as,
in particular part of
the cell cycle, or at a particular stage where particular genes are being
expressed) where cells
are being used, culture conditions (including cell media, substrates, oxygen,
carbon dioxide,
glucose and other sugar substrates, serum, growth factors, etc.).
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[0146] The present disclosure also provides for the use of cell lines
that have enhanced
properties for growth, reduced dependency upon expensive factors that are
present in media,
produce higher yields of proteins, and produce higher titers of vector
particles. For example it
has recently been reported HEK 293 cells have a specific increased expression
of cellular
receptors and by adding the specific ligands to the medium of the cells, they
demonstrated
increase proliferation potential (Allison et at., Bioprocess International,
2005, 3(1): 38-45). A
preferred aspect is a plurality of Lentiviral vectors expressing an optimized
combination of
ligand proteins that are of relevance to HEK 293 cells after which the cells
are then sorted by
high throughput methods to isolate a clone of HEK 293 cells that contains
multiple copies of
lentiviral vectors. These cells contain a combination of HIV vectors that
express different but
also multiple copies of the ligand genes that are contained in the HIV
vectors. The ligand genes
could be codon optimized or mutations added to further increase their
expression. A preferred
combination is to have multiple copies of the ligand proteins expressed in the
final isolated
clonal cell that could then have multiple uses. It could be used for protein
or antibody (including
monoclonal, humanized, single-chain) production. It could also be used for the
production of
a vector such as a lentiviral vector, but not limited to a lentiviral vector.
Other vectors such as
adeno and adeno-associated vectors, murine retroviral vectors, SV40 vectors
and other vectors
could just as easily be produced from this now optimized cell line. A list of
the receptors and
their ligands that show increased expression/activity in HEK 293 cells,
includes, e.g., AXL
receptor (gasf3); EGF receptor (EGF), chemokine receptor (fractalline); PDGF
receptor, beta
(PDGF); IL-15R-alpha; IL- 2R-alpha; chemokine receptor 2 (MCP1); IL-2R, gamma;
IL-1R-1;
CSF-I receptor; oncostatin receptor; IL-4R; vitamin D3 receptor; neuropilin 1
(VEGF);
macrophage stimulating receptor 1 (MSP); NGF-R; PDGFR-alpha receptor; IL-11-R,
e.g.,
alpha; IL- 10-R, e.g., beta; FGF-R-4 (aFGF); BMP receptor, e.g., type II (BMP-
2); TGF-R,
e.g., beta receptor II (TGF-beta); FGF-R-I (bFGF); chemokine receptor 4
(SFDIa); interferon
gamma receptor 1 and 2. See, BioProcess International, January 2005. Table 1,
"Growth
factor/cytokine receptors expressed by HEK-293." Such cells will have higher
protein and
vector production potential and will be less dependent upon the presence of
the ligand factors
to be present in the medium since the cells themselves will be producing the
factors and
secreting them into the medium.
[0147] For other cell types, such as CHO cells, other receptor-ligand
combinations may be
important. For example the insulin growth factor receptor I, insulin growth
factor and insulin
are thought to have anti-apoptotic activity in cells. A plurality lentiviral
vectors could be
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constructed so that the insulin growth factor receptor (I or II), insulin
growth factor (I or II),
insulin and the target protein for production are all contained in the vector
for transduction of
production cells, such as CHO cells, and an appropriate clone selected,
preferably using high-
throughput methods, to select the clone showing very high production of the
target protein. The
optimal clone may not be a cell that highly expresses all the engineered genes
or inhibitors of
gene expression, rather an optimal expression level of each of the genes,
which for some may
be a low level of expression. The value of the lentiviral vector system and
using a plurality of
lentiviral vectors to engineer such cell lines is that there is a random or
stochastic distribution
of each vector copy number in the population of cells transduced with the
lentiviral vector
mixture, and therefore, by varying the amount of each vector in the mixture,
the number of
copies of each individual second gene or inhibitory sequence can be optimized.
A preferred
combination of vectors and secondary gene or gene inhibitory sequences is that
each lentiviral
vector expresses the protein of interest for production and optionally in
addition, at least one
RNAi or gene that further promotes protein yield, or vector yield, either
directly, or indirectly
by affecting the viability or some aspect of the producing cell. However, it
may also be
beneficial to have at least one lentiviral vector that only expresses the
secondary genes or
inhibitors of gene expression in order to increase the effect of these
secondary sequences.
Exemplary embodiments
[0148] Embodiment 1. A method of making a stable viral vector producer
cell line, said
method comprising:
a. introducing into a population of cells a viral vector genome construct
encoding a
gene of interest (GOT) and one or more viral accessory constructs encoding one
or more
viral accessory proteins;
b. producing a population of transgenic cells comprising integrated or
episomal
sequences encoding said GOT and said one or more viral accessory proteins;
c. selecting from said population of transgenic cells a cell clone
producing a desired
viral titer; and
d. generating from said cell clone a stable viral vector producer cell line,
wherein the introduction of said one or more accessory constructs occurs
concurrently.
[0149] Embodiment 2. A method of making a stable viral vector producer cell
line, said
method comprising:
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a. introducing into a population of cells a viral vector genome construct
encoding a
gene of interest (GOT) and one or more viral accessory constructs encoding one
or more
viral accessory proteins;
b. producing a population of transgenic cells comprising integrated or
episomal
sequences encoding said GOT and said one or more viral accessory proteins;
c. selecting from said population of transgenic cells a cell clone
producing a desired
viral titer; and
d. generating from said cell clone a stable viral vector producer cell line,
wherein the introduction of said one or more accessory constructs occurs via
one or
more sequential steps with no intervening cell culturing.
[0150] Embodiment 3. The method of Embodiment 1 or 2, wherein said
transgenic cells
comprise polyclonal cells.
[0151] Embodiment 4. The method of Embodiment 1 or 2, wherein said
selecting
further comprises polyclonal to monoclonal selection of said transgenic cells.
[0152] Embodiment 5. The method of Embodiment 4, wherein said polyclonal to
monoclonal selection comprises limiting dilution, single cell sorting, single
cell selection, or a
combination thereof.
[0153] Embodiment 6. The method of any one of Embodiments 1 to 5,
wherein said
generation of said stable viral vector producer cell line occurs by expansion
of said selected
cell clone.
[0154] Embodiment 7. The method of Embodiment 1 or 2, wherein said
method further
comprises storing said selected cell line by cryopreservation.
[0155] Embodiment 8. The method of Embodiment 7, wherein said method
further
comprises expanding cells from said cryopreserved cell line to produce viral
vectors.
[0156] Embodiment 9. The method of Embodiment 1 or 2, wherein said method
further
comprises quantifying the level of said viral vector genome and said one or
more accessory
proteins in said selected cell clone, said generated cell line, or both.
[0157] Embodiment 10. The method of Embodiment 1 or 2, wherein said
method further
comprises determining the stoichiometric ratio of viral vector genome RNA and
one or more
accessory proteins in said selected cell clone, said generated cell line, or
both.
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[0158] Embodiment 11. The method of Embodiment 1 or 2, wherein said
method further
comprises determining an integration profile of said selected cell clone, said
generated cell line,
or both.
[0159] Embodiment 12. The method of Embodiment 1 or 2, wherein said
method further
comprises harvesting viral vector from said selected cell clone, said
generated cell line, or both.
[0160] Embodiment 13. The method of Embodiment 1 or 2, wherein said
method further
comprises determining a viral titer of said selected cell clone, said
generated cell line, or both.
[0161] Embodiment 14. The method of Embodiment 13, wherein determining
said viral
titer comprises physical titration, functional titration, or both.
[0162] Embodiment 15. The method of Embodiment 13, wherein determining said
viral
titer is determined by assaying for viral nucleic acid via an assay selected
from the group
consisting of PCR, RT-PCR, and quantitative detection by blot hybridization,
or assaying for
a viral protein via immunoassay.
[0163] Embodiment 16. The method of Embodiment 14, wherein said method
further
comprises determining an infectivity of said viral titer of said selected cell
clone or said
generated cell line.
[0164] Embodiment 17. The method of Embodiment 1 or 2, wherein said
viral vector
producer cell line produces a target-specific viral vector.
[0165] Embodiment 18. The method of Embodiment 1 or 2, wherein said
viral vector
producer cell line produces a viral vector derived from a retrovirus.
[0166] Embodiment 19. The method of Embodiment 1 or 2, wherein said
viral vector
producer cell line produces a viral vector derived from a lentivirus.
[0167] Embodiment 20. The method of Embodiment 1 or 2, wherein said
viral vector
producer cell line produces a viral vector derived from a herpesvirus.
[0168] Embodiment 21. The method of Embodiment 1 or 2, wherein said viral
vector
producer cell line produces a viral vector derived from an adenovirus.
[0169] Embodiment 22. The method of Embodiment 1 or 2, wherein said
viral vector
producer cell line produces a viral vector derived from an adeno-associated
virus.
[0170] Embodiment 23. The method of Embodiment 1 or 2, wherein said
viral vector
producer cell line produces a viral vector comprising one or more capsid
proteins.
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[0171] Embodiment 24. The method of Embodiment 23, wherein said one or
more capsid
proteins are heterologous.
[0172] Embodiment 25. The method of Embodiment 23, wherein said one or
more capsid
proteins are genetically modified.
[0173] Embodiment 26. The method of Embodiment 23, wherein said one or more
capsid proteins are chemically modified.
[0174] Embodiment 27. The method of Embodiment 1 or 2, wherein said
viral vector
producer cell line produces a viral vector comprising one or more envelope
proteins.
[0175] Embodiment 28. The method of Embodiment 27, wherein said one or
more
envelope proteins are heterologous.
[0176] Embodiment 29. The method of Embodiment 1 or 2, wherein said
viral vector
genome construct comprises one or more elements selected from the group
consisting of a 5'
long terminal repeat, a 3' long terminal repeat, a packaging signal, and a
central polypurine
tract.
[0177] Embodiment 30. The method of Embodiment 1 or 2 wherein said viral
vector
genome construct does not comprise a 5' long terminal repeat, a 3' long
terminal repeat, a
packaging signal, or a central polypurine tract.
[0178] Embodiment 31. The method of Embodiment 29, wherein said 5' long
terminal
repeat is chimeric.
[0179] Embodiment 32. The method of Embodiment 1 or 2, wherein said viral
vector
genome construct comprises a self-inactivating long terminal repeat.
[0180] Embodiment 33. The method of Embodiment 1 or 2, wherein said
viral vector
genome construct comprises one or more selectable or reporter elements.
[0181] Embodiment 34. The method of Embodiment 33, wherein said one or
more
selectable or reporter elements is a reporter gene, an epitope tag, or both.
[0182] Embodiment 35. The method of Embodiment 33, wherein said one or
more
selectable or reporter elements is selected or detected by luminescence,
absorbance,
fluorescence, antibiotics, antigen-antibody interactions, or a combination
thereof
[0183] Embodiment 36. The method of Embodiment 1 or 2, wherein said
viral vector
genome construct comprises a promoter and a polyadenylation sequence.
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[0184] Embodiment 37. The method of Embodiment 36, wherein said promoter
of said
viral vector genome construct is constitutive or inducible.
[0185] Embodiment 38. The method of Embodiment 36, wherein said promoter
of said
viral vector genome construct is synthetic.
[0186] Embodiment 39. The method of Embodiment 1 or 2, wherein said viral
vector
genome construct comprises an insulator sequence.
[0187] Embodiment 40. The method of Embodiment 1 or 2, wherein said
viral vector
genome construct comprises a concatemer.
[0188] Embodiment 41. The method of Embodiment 40, wherein said
concatemer
comprises multiple copies of an expression cassette encoding said GOT.
[0189] Embodiment 42. The method of Embodiment 40, wherein said
concatemer
comprises one or more expression cassettes encoding a transcription factor.
[0190] Embodiment 43. The method of Embodiment 40, wherein said
concatemer
comprises one or more expression cassettes encoding an antibiotic selection
gene.
[0191] Embodiment 44. The method of Embodiment 1 or 2, wherein said one or
more
viral accessory constructs comprises a promoter and a polyadenylation
sequence.
[0192] Embodiment 45. The method of Embodiment 1 or 2, wherein said one
or more
viral accessory constructs comprises an enhancer sequence.
[0193] Embodiment 46. The method of Embodiment 1 or 2, wherein said one
or more
viral accessory constructs comprises an insulator sequence.
[0194] Embodiment 47. The method of Embodiment 44, wherein said promoter
of said
one or more viral accessory constructs comprises a promoter/enhancer.
[0195] Embodiment 48. The method of Embodiment 44, wherein said promoter
of said
one or more viral accessory constructs is a synthetic promoter.
[0196] Embodiment 49. The method of Embodiment 44, wherein said promoter of
said
one or more viral accessory constructs is selected from the group consisting
of an inducible,
constitutive, switched, recombined, or disrupted/edited promoter.
[0197] Embodiment 50. The method of Embodiment 1 or 2, wherein said one
or more
viral accessory proteins are fusion proteins.
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[0198] Embodiment 51. The method of Embodiment 1 or 2, wherein said one
or more
viral accessory constructs comprises one or more expression cassettes.
[0199] Embodiment 52. The method of Embodiment 51, wherein said
expression cassette
is a monocistronic expression cassette or a polycistronic expression cassette.
[0200] Embodiment 53. The method of Embodiment 52, wherein said
polycistronic
expression cassettes further comprises one or more viral skip sequences,
internal ribosome
entry site elements, or both.
[0201] Embodiment 54. The method of Embodiment 53, wherein said viral
skip
sequences are selected from the group consisting of P2A, T2A, E2A, and F2A.
[0202] Embodiment 55. The method of Embodiment 1 or 2, wherein said one or
more
viral accessory proteins comprise sequences encoding structural viral
proteins, regulatory viral
proteins, or both.
[0203] Embodiment 56. The method of Embodiment 55, wherein said
structural proteins
and/or regulatory proteins are selected from the group consisting of Gag, Pol,
Rev, Env, Tat,
Nef, Vpr, Vif, Vpu, and Vpx.
[0204] Embodiment 57. The method of Embodiment 50, wherein said viral
accessory
constructs comprise sequences encoding a partial viral accessory protein.
[0205] Embodiment 58. The method of Embodiment 57, wherein said partial
viral
accessory protein comprises one or more viral accessory protein domains.
[0206] Embodiment 59. The method of Embodiment 57, wherein said one or more
viral
accessory protein domains is selected from the group consisting of CA, MA, NC,
p6, SP1, RT,
IN, PR, and DU.
[0207] Embodiment 60. The method of any one of Embodiments 55 to 59,
wherein a
sequence encoding said one or more viral accessory proteins or domains
comprises a wild-type
sequence, a mutated sequence, a codon optimized sequence, or a combination
thereof.
[0208] Embodiment 61. The method of any one of Embodiments 55 to 60,
wherein said
one or more viral accessory proteins or viral accessory protein domains are
introduced via
separate expression cassettes.
[0209] Embodiment 62. The method of Embodiment 56, wherein said env
protein
comprises a bioengineered chimeric envelope protein.
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[0210] Embodiment 63. The method of Embodiment 56, wherein said env
protein is
linked to an antibody or to a ligand.
[0211] Embodiment 64. The method of Embodiment 56, wherein said env
protein is
derived from human immunodeficiency virus.
[0212] Embodiment 65. The method of Embodiment 56, wherein said env protein
is
derived from a virus selected from the group consisting of Vesiculovirus,
Gammaretrovirus,
and Morbillivirus.
[0213] Embodiment 66. The method of Embodiment 65, wherein said
Vesiculovirus is
selected from the group consisting of vesicular stomatitis New Jersey virus
(VSV-NJ),
.. vesicular stomatitis Indiana virus (VSV-IN), and strains derived therefrom.
[0214] Embodiment 67. The method of Embodiment 65, wherein said
Gammaretrovirus
is selected from the group consisting of gibbon ape leukemia virus, feline
leukemia virus, and
derivatives thereof.
[0215] Embodiment 68. The method of Embodiment 65, wherein said
Morbillivirus is
.. selected from the group consisting of measles virus and derivatives
thereof.
[0216] Embodiment 69. The method of Embodiment 1 or 2, wherein said
introducing step
comprises a chemical, biological, or physical step.
[0217] Embodiment 70. The method of Embodiment 1 or 2, wherein said
introducing step
comprises an optical method, a magnetic method, a biolistic method, a polymer-
based method,
a liposome-based method, a nanoparticle-based method, or a combination
thereof.
[0218] Embodiment 71. The method of Embodiment 1 or 2, wherein said
introducing step
comprises a transduction.
[0219] Embodiment 72. The method of Embodiment 1 or 2, wherein said
introducing step
comprises a transfection.
[0220] Embodiment 73. The method of Embodiment 69, wherein said chemical
introducing step comprises the use of a cationic polymer, calcium phosphate,
cationic lipid, or
a combination thereof.
[0221] Embodiment 74. The method of Embodiment 69, wherein said
biological
introducing step comprises introduction via a retrovirus, lentivirus,
transposon, TALEN, Zinc
.. Finger nuclease, meganuclease, transposase, CRISPR-related nuclease, or
recombinase.
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[0222] Embodiment 75. The method of Embodiment 74, wherein said
recombinase
comprises Cre-recombinase or Flippase recombinase.
[0223] Embodiment 76. The method of Embodiment 69, wherein said physical
introducing step is selected from the group consisting of electroporation,
sonoporation,
mechanoporation, and photoporation.
[0224] Embodiment 77. The method of Embodiment 1 or 2, wherein said
integrated
sequences exhibit random integration.
[0225] Embodiment 78. The method of Embodiment 1 or 2, wherein said
integrated
sequences exhibit site-specific integration.
[0226] Embodiment 79. The method of Embodiment 1 or 2, wherein said stable
viral
vector producer cell line is in a cell culture that comprises a volume of
medium.
[0227] Embodiment 80. The method of Embodiment 1 or 2, wherein said
stable viral
vector producer cell line is adapted for adherent culturing or culturing in
suspension.
[0228] Embodiment 81. The method of Embodiment 1 or 2, wherein said
stable viral
vector producer cell line is cultured in a serum-supplemented or serum-free
medium.
[0229] Embodiment 82. The method of Embodiment 1 or 2, wherein said
stable viral
vector producer cell line is immortalized.
[0230] Embodiment 83. The method of Embodiment 1 or 2, wherein said
stable viral
vector producer cell line is eukaryotic.
[0231] Embodiment 84. The method of Embodiment 1 or 2, wherein said stable
viral
vector producer cell line is mammalian.
[0232] Embodiment 85. The method of Embodiment 1 or 2, wherein said
stable viral
vector producer cell line is human.
[0233] Embodiment 86. The method of Embodiment 1 or 2, wherein said
stable viral
vector producer cell line is a HEK293 cell or a derivative thereof.
[0234] Embodiment 87. The method of Embodiment 86, wherein said HEK293
cell is a
HEK293T cell.
[0235] Embodiment 88. The method of Embodiment 1 or 2, wherein said
stable viral
vector producer cell line produces psuedotyped viral particles.
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[0236] Embodiment 89. The method of Embodiment 88, wherein said
pseudotyped viral
particles comprise one or more envelope proteins of a virus selected from the
group consisting
of Vesiculovirus, Gammaretrovirus, and Morbillivirus.
[0237] Embodiment 90. The method of Embodiment 1 or 2, wherein said
stable viral
vector producer cell line produces mosaic viral particles.
[0238] Embodiment 91. The method of Embodiment 1 or 2, wherein said
stable viral
vector producer cell line produces chimeric viral particles.
[0239] Having now generally described the disclosure, the same will be
more readily
understood through reference to the following examples that are provided by
way of
illustration, and are not intended to be limiting of the present disclosure,
unless specified.
EXAMPLES
Example 1:
[0240] As an illustration of the concept described here, an HIV-based
lentiviral vector
producer cell line is produced (Figure 1). Figure 2 summarizes a
representative work flow of
generating a cell clone with stable introduction of various construct
elements, which can
subsequently be expanded, cryopreserved, and banked.
Example 2:
[0241] An experiment is conducted to test two different vector
constructs using a battery
of different component ratios to demonstrate that each vector produces best
titer using a
.. different ratio. The accessory genes are delivered using standard packing
plasmids one each
for gag/pol, rev, and env. The vector constructs (a.k.a. GOT, for gene of
interest) each deliver
a green fluorescent protein reporter cassette which is used to determine
vector infectivity by
standard titration assay. The first GOT construct is called GFP and delivers a
simple, single
expression cassette in a 4.2kb provirus (Figure 3). The second GOT is a more
complex and
clinically relevant construct of about 10kb containing the GFP reporter
cassette, a 3.3 kb
structural element called the locus control region (LCR), and the human beta
globin cassette
which expresses from its native promoter in the antisense orientation allowing
the
incorporation of two introns which are spliced out of the final mRNA (Figure
3). This
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construct (shown in Figure 3as Globin-LCR-GFP) is known to produce lower
titers than GFP.
Both constructs are tested following the vector production protocol summarized
below.
[0242] HEK293 packaging cell preparation: To a log phase expanding shake
flask of
HEK293 cells, remove a sample and count, then dilute the cells to 3.5e6
cells/ml by adding
fresh media. Incubate overnight while shaking at 37 C, 8% CO2. Sample and
count the cells,
diluting with fresh media to bring to 4.7e6 cells/ml. Add 25.5m1 of cell
suspension to a new
125m1 shake flask for each condition. Add 1.5m1 of LV-MAX Supplement and swirl
to mix.
Return to incubator while preparing DNA transfection mixes.
[0243] DNA transfection: For each condition, add the indicated volume of
each of the 4
plasmids to a labeled 15m1 conical tube and dilute with Opti-MEM media to
bring to 1.5m1
total (per Table 2). Swirl/tap to mix. For each condition, add 180u1 of LV-MAX
reagent and
1.32m1 of Opti-MEM media to a second labeled 15m1 conical tube. Swirl/tap to
mix. Add the
diluted plasmid DNA to the diluted LV-MAX Transfection Reagent and gently
pipette up and
down to mix. Allow the transfection mixture to incubate for 10 mins at room
temperature.
Retrieve the target cells from the incubator and slowly transfer the
transfection mix to the
shaker flask for each condition, swirl gently to mix, and return to the shaker
incubator for 48-
55 hours.
[0244] Vector Harvest: Transfer cultures to a 50m1 conical tube and
centrifuge at 1500xg
for 5 minutes at room temperature. Transfer supernatant to a 60m1 syringe
fitted with a 0.45
micron PES syringe filter and apply gentle pressure to slowly filter the
supernatants into a
new 50m1 conical tube. Aliquot the clarified vector preparations into labeled
15m1 conical
tubes, between 3 and 5m1 per tube, snap freeze over dry ice, and then store at
-20 C until
ready to use.
[0245] Target Cell Preparation: To a log-phase growing culture of SupT1
cells, remove a
sample and count, then dilute the cells to 1e6 cells/ml with fresh media. Add
1000x
protamine sulfate to make the final cell suspension 2x (2u1 per every lml of
cells). Plate the
cell mixture into 96 well plates, 1 row for every vector prep as indicated.
[0246] Vector Preparation: Thaw all vector lots to be tested by allowing
to incubate at
room temperature until fully thawed. Label four 15m1 conical tubes 1:2, 1:10,
1:20, 1:100. To
the 1:2 tube add 2m1 of thawed vector and 2m1 of fresh SupT I media, invert 2-
3 times to mix.
To the 1:10 tube add lml of thawed vector and 9m1 of fresh SupT I media,
invert 2-3 times to
mix. To the 1:20 tube add lml of the 1:2 dilution and 9m1 of fresh SupT I
media, invert 2-3
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times to mix. To the 1:100 tube add lml of the 1:10 dilution and 9m1 of fresh
SupT1 media,
invert 2-3 times to mix. Add the diluted vectors to the cell culture plate
100u1 per well, each
dilution in triplicate, one row per vector (8 rows per plate allows 8 vectors
to be tested in this
manner). Incubate the titer plates for 3 days at 37 C, 5% CO2.
[0247] Titer Determination: Use flow cytometry to determine the %GFP
positivity versus
an untransduced SupT1 control for each well of the titer plate. Determine
titer per well using
the following formula: (1e5 cells * %GFP+)/(100u1* dilution factor)=titer in
tu/ml. For all
samples of a given vector prep with a %GFP+ between 1-10%, determine the
arithmetic
mean. That mean value is the observed titer for that prep.
[0248] The Globin-LCR-GFP construct shows lower titers than the GFP vector.
The
optimal titer observed for the GFP vector is about 1e7 tu/ml using condition
16 (ratio of
9:1:1:9), the next optimal titer is 8e6 tu/ml using condition 4 (ratio of
3:1:1:3). The optimal
titer for Globin-LCR-GFP is 1e6 tu/ml using condition 12 (ratio of 9:1:1:6)
and the second
most optimal is condition 6 (ratio of 6:1:1:3) with a titer of 2e5 tu/ml
(Figure 4)
[0249] This experiment demonstrates that for different GOT constructs,
different ratios of
constituent elements are required to achieve optimal vector particle
infectious titer. This
experiment also demonstrates that by using an array of starting ratios, it is
unnecessary to
know ahead of time what the optimal ratio will be, as it can be subsequently
determined
empirically. This experiment shows that manufacturing a stable production
system using a
.. fixed ratio of components is unlikely to produce optimal titers for any
possible GOT, and only
likely to work well using a narrow spectrum of constructs. This experiment
provides an
exemplary embodiment described here which allows for a range of possible
vector ratios and
combinations to occur, then empirically testing the resulting lines to find
the optimal
producer cell clone.
30
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Table 2: Exemplary plasmid ratios used to test for optimal combinations for
distinct test
constructs.
RATIO Plasmid quantity (u.g)
Condition gag/pol rev env GOT gag/pol rev env
GOT
1 1 1 1 1 18.75 18.75 18.75
18.75
2 3 1 1 1 37.50 12.50 12.50
12.50
3 1 1 1 3 12.50 12.50 12.50
37.50
4 3 1 1 3 28.13 9.38 9.38
28.13
6 1 1 1 50.00 8.33 8.33 8.33
6 6 1 1 3 40.91 6.82 6.82
20.45
7 1 1 1 6 8.33 8.33 8.33
50.00
8 3 1 1 6 20.45 6.82 6.82
40.91
9 6 1 1 6 32.14 5.36 5.36
32.14
9 1 1 1 56.25 6.25 6.25 6.25
11 9 1 1 3 48.21 5.36 5.36
16.07
12 9 1 1 6 39.71 4.41 4.41
26.47
13 1 1 1 9 6.25 6.25 6.25
56.25
14 3 1 1 9 16.07 5.36 5.36
48.21
6 1 1 9 26.47 4.41 4.41 39.71
16 9 1 1 9 33.75 3.75 3.75
33.75
44
